The present invention is directed to compounds of the formula 
and pharmaceutically acceptable salts thereof. As used in formula I, and throughout the specification, the symbols have the following meanings:
R1 and R2 are independently hydrogen, fluorine or alkyl;
R3 is aryl or heteroaryl
R4 is alkyl, cycloalkyl, aryl, cycloalkylalkyl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl; or
CO-alkyl, CO-cycloalkyl, CO-aryl, CO-alkyl-cycloalkyl, CO-alkyl-aryl, CO-heteroaryl, CO-alkyl-heteroaryl, CO-heterocycloalkyl, CO-alkyl-heterocycloalkyl; or
CONH-alkyl, CONH-cycloalkyl, CONH-aryl, CONH-alkyl-cycloalkyl, CONH-alkyl-aryl, CONH-heteroaryl, CONH-alkyl-heteroaryl, CONH-heterocycloalkyl, CONH-alkyl-heterocycloalkyl; or
COO-alkyl, COO-cycloalkyl, COO-aryl, COO-alkyl-cycloalkyl, COO-alkyl-aryl, COO-heteroaryl, COO-alkyl-heteroaryl, COO-heterocycloalkyl, COO-alkyl-heterocycloalkyl; or
SO2-cycloalkyl, SO2-aryl, S2-alkyl-cycloalkyl, SO2-alkyl-aryl, SO2-heteroaryl, SO2-alkyl-heteroaryl, SO2-heterocycloalkyl, SO2-alkyl-heterocycloalkyl; or
C(NCN)NH-alkyl, C(NCN)NH-cycloalkyl, C(NCN)NH-aryl, C(NCNNH)-alkyl-cycloalkyl, C(NCN)NH-alkyl-aryl, C(NCN)NH-heteroaryl, C(NCN)NH-alkyl-heteroaryl, C(NCN)NH-heterocycloalkyl, C(NCN)NH-alkyl-heterocylcoalkyl; or
C(NNO2)NH-alkyl, C(NNO2)NH-cycloalkyl, C(NNO2)NH-aryl, C(NNO2)NH-alkyl-cycloalkyl, C(NNO2)NH-alkyl-aryl, C(NNO2)NH-heteroaryl, C(NNO2)NH-alkyl-heteroaryl, C(NNO2)NH-heterocyloalkyl, C(NNO2)NH-alkyl-heterocycloalkyl; or
C(NH)NH-alkyl, C(NH)NH-cycloalkyl, C(NH)NH-aryl, C(NH)NH-alkyl-cycloalkyl, C(NH)NH-alkyl-aryl, C(NH)NH-heteroaryl, C(NH)NH-alkyl-heteroaryl, C(NH)NH-heterocycloalkyl, C(NH)NH-alkyl-heterocycloalkyl; or
C(NH)NHCO-alkyl, C(NH)NHCO-cycloalkyl, C(NH)NHCO-aryl, C(NH)NHCO-alkyl-cycloalkyl, C(NH)NHCO-alkyl-aryl, C(NH)NHCO-heteroaryl, C(NH)NHCO-alkyl-heteroaryl, C(NH)NHCO-heterocylcloalkyl, C(NH)NHCO-alkyl-heterocycloalkyl; or
C(NOR6)NH-alkyl, C(NOR6)NH-cycloalkyl, C(NOR6)NH-aryl, C(NOR6)NH-alkyl-cycloalkyl, C(NOR6)NH-alkyl-aryl, C(NOR6)NH-heteroaryl, C(NOR6)NH-alkyl-heteroaryl, C(NOR6)NH-heterocylcoalkyl, C(NOR6)NH-alkyl-heterocycloalkyl;
R5 is hydrogen or alkyl;
R6 is hydrogen, alkyl, cycloalkyl, aryl, cycloalkylakyl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl or heterocycloalkylalkyl;
m is an integer of 0 to 2; and
n is an integer of 1 to 3.
The compounds of formula I are protein kinase inhibitors and are useful in the treatment and prevention of proliferative diseases, for example, cancer, inflammation and arthritis. They may also be useful in the treatment of neurodegenerative diseases such as Alzheimer""s disease, cardiovascular diseases, viral diseases and fungal diseases.
The present invention provides for compounds of formula I, pharmaceutical compositions employing such compounds and for methods of using such compounds.
Listed below are definitions of various terms used to describe the compounds of the instant invention. These definitions apply to the terms as they are used throughout the specification (unless they are otherwise limited in specific instances) either individually or as part of a larger group.
It should be noted that any heteroatom with unsatisfied valances is assumed to have the hydrogen atom to satisfy the valances.
Carboxylate anion refers to a negatively charged group xe2x80x94COOxe2x88x92.
The term xe2x80x9calkylxe2x80x9d or xe2x80x9calkxe2x80x9d refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 12 carbon atoms unless otherwise defined. An alkyl group is an optionally substituted straight, branched or cyclic saturated hydrocarbon group. When substituted, alkyl groups may be substituted with up to four substituent groups, R as defined, at any available point of attachment. When the alkyl group is said to be substituted with an alkyl group, this is used interchangeably with xe2x80x9cbranched alkyl groupxe2x80x9d. Exemplary unsubstituted such groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. Exemplary substituents may include but are not limited to one or more of the following groups: halo (such as F, Cl, Br, I), haloalkyl (such as CCl3 or CF3), alkoxy, alkylthio, hydroxy, carboxy (xe2x80x94COOH), alkyloxycarbonyl (xe2x80x94C(O)R), alkylcarbonyloxy (xe2x80x94OCOR), amino (xe2x80x94NH2), carbamoyl (xe2x80x94NHCOORxe2x80x94 or xe2x80x94OCONHRxe2x80x94), urea (xe2x80x94NHCONHRxe2x80x94) or thiol (xe2x80x94SH). Alkyl groups as defined may also comprise one or more carbon to carbon double bonds or one or more carbon to carbon triple bonds.
The term xe2x80x9calkenylxe2x80x9d refers to a hydrocarbon radical straight, branched or cyclic containing from 2 to 12 carbon atoms and at least one carbon to carbon double bond.
The term xe2x80x9calkynylxe2x80x9d refers to a hydrocarbon radical straight, branched or cyclic containing from 2 to 12 carbon atoms and at least one carbon to carbon triple bond.
Cycloalkyl is a specie of alkyl containing from 3 to 15 carbon atoms, without alternating or resonating double bonds between carbon atoms. It may contain from 1 to 4 rings. Exemplary unsubstituted such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, etc. Exemplary substituents include one or more of the following groups: halogen, alkyl, alkoxy, alkyl hydroxy, amino, nitro, cyano, thiol and/or alkylthio.
The terms xe2x80x9calkoxyxe2x80x9d or xe2x80x9calkylthioxe2x80x9d, as used herein, denote an alkyl group as described above bonded through an oxygen linkage (xe2x80x94Oxe2x80x94) or a sulfur linkage (xe2x80x94Sxe2x80x94), respectively.
The term xe2x80x9calkyloxycarbonylxe2x80x9d, as used herein, denotes an alkoxy group bonded through a carbonyl group. An alkoxycarbonyl radical is represented by the formula: xe2x80x94C(O)OR, where the R group is a straight or branched C1-6 alkyl group.
The term xe2x80x9calkylcarbonylxe2x80x9d refers to an alkyl group bonded through a carbonyl group.
The term xe2x80x9calkylcarbonyloxyxe2x80x9d, as used herein, denotes an alkylcarbonyl group which is bonded through an oxygen linkage.
The term xe2x80x9carylalkylxe2x80x9d, as used herein, denotes an aromatic ring bonded to an alkyl group as described above.
The term xe2x80x9carylxe2x80x9d refers to monocyclic or bicyclic aromatic rings, e.g. phenyl, substituted phenyl and the like, as well as groups which are fused, e.g., napthyl, phenanthrenyl and the like. An aryl group thus contains at least one ring having at least 6 atoms, with up to five such rings being present, containing up to 22 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms or suitable heteroatoms. Aryl groups may optionally be substituted with one or more groups including, but not limited to halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, nitro, trifluoromethyl, amino, cycloalkyl, cyano, alkyl S(O)m (m=0, 1, 2), or thiol.
The term xe2x80x9cheteroarylxe2x80x9d refers to a monocyclic aromatic hydrocarbon group having 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing at least one heteroatom, O, S, or N, in which a carbon or nitrogen atom is the point of attachment, and in which one or two additional carbon atoms is optionally replaced by a heteroatom selected from O or S, and in which from 1 to 3 additional carbon atoms are optionally replaced by nitrogen heteroatoms, said heteroaryl group being optionally substituted as described herein. Exemplary heteroaryl groups include the following: thienyl, furyl, pyrrolyl, pyridinyl, imidazolyl, pyrrolidinyl, piperidinyl, thiazolyl, oxazolyl, triazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyrazinyl, pyridazinyl, pyrimidinal, triazinylazepinyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzoxadiazolyl, benzofurazanyl and tetrahydropyranyl. Exemplary substituents include one or more of the following: halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, trifluoromethyl, cycloalkyl, nitro, cyano, amino, alkylS(O)m (m=0, 1, 2), or thiol.
The term xe2x80x9cheteroaryliumxe2x80x9d refers to heteroaryl groups bearing a quaternary nitrogen atom and thus a positive charge.
The term xe2x80x9cheterocycloalkylxe2x80x9d refers to a cycloalkyl group (nonaromatic) in which one of the carbon atoms in the ring is replaced by a heteroatom selected from O, S or N, and in which up to three additional carbon atoms may be replaced by said heteroatoms.
The term xe2x80x9cquaternary nitrogenxe2x80x9d refers to a tetravalent positively charged nitrogen atom including, e.g. the positively charged nitrogen in a tetraalkylammonium group (e.g. tetramethylammonium, N-methylpyridinium), the positively charged nitrogen in protonated ammonium species (e.g. trimethylhydroammonium, N-hydropyridinium), the positively charged nitrogen in amine N-oxides (e.g. N-methyl-morpholine-N-oxide, pyridine-N-oxide), and the positively charged nitrogen in an N-amino-ammonium group (e.g. N-aminopyridinium).
The term xe2x80x9cheteroatomxe2x80x9d means O, S or N, selected on an independent basis.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refers to chlorine, bromine, fluorine or iodine.
When a functional group is termed xe2x80x9cprotectedxe2x80x9d, this means that the group is in modified form to preclude undesired side reactions at the protected site. Suitable protecting groups for the compounds of the present invention will be recognized from the present application taking into account the level of skill in the art, and with reference to standard textbooks, such as Greene, T. W. et al., Protective Groups in Organic Synthesis, Wiley, N.Y. (1991).
Suitable examples of salts of the compounds according to the invention with inorganic or organic acids are hydrochloride, hydrobromide, sulfate, phosphate. Salts which are unsuitable for pharmaceutical uses but which can be employed, for example, for the isolation or purification of free compounds I or their pharmaceutically acceptable salts, are also included.
All stereoisomers of the compounds of the instant invention are contemplated, either in admixture or in pure or substantially pure form. The definition of the compounds according to the invention embraces all possible stereoisomers and their mixtures. It very particularly embraces the racemic forms and the isolated optical isomers having the specified activity. The racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates by conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.
It should be understood that solvates (e.g., hydrates) of the compounds of formula I are also within the scope of the present invention. Methods of solvation are generally known in the art. Accordingly, the compounds of the instant invention may be in the free or hydrate form, and may be obtained by methods exemplified by the following schemes. 
As illustrated in Scheme 1, compounds of formula I where X is S are prepared by reacting 2-aminothiazole (II) with bromine in the presence of sodium or potassium thiocyanate to obtain a thiocyanated aminothiazole, specifically 5-thiocyanatoaminothiazole (III). Compound III is then reacted with R4xe2x80x94L, where L is a leaving group such as a halogen, in the presence of a base such as triethylamine to provide a 5-thiocyanatothiazole intermediate (IV), where R4 is as defined in the specification. The intermediate (IV) is then reduced to a thiol (V) using reducing agents such as dithiothreitol (DTT), sodium borohydride, zinc or other known reducing agents. Compound (V) is then reacted with alkyl, aryl or heteroaryl halides, such as R3 (CR1R2)nxe2x80x94L, where L is a leaving group such as a halogen, in the presence of a base such as potassium carbonate to obtain compounds of formula I. The steps of reducing the thiocyanothiazole intermediate (IV) to the thiol (V), and the reaction of the reduced thiol (V) to provide compounds of formula I where X is S, may be carried out sequentially without purification. 
In Scheme 2, 5-thioacetyl-2-acetylaminothiazole of structure VI is reacted with an alkoxide such as potassium t-butoxide in alcohol or THF solvent and the resulting thiol is reacted in situ with a group of formula R3(CR1R2)nxe2x80x94L (where L is a leaving group, such as a halogen) such as 2-halomethyloxazole (VII) to provide a compound such as formula VIII, wherein R1 and R2 are hydrogen, and R6 is acetyl. The 2-halomethyloxazole compounds of formula VII may be prepared using several synthetic routes known in the art. Chem. Pharm. Bull. 30, 1865 (1982); Bull. Chem. Soc. Japan (52, 3597 (1979); JCS Chem. Comm. 322 (1981); Comprehensive Heterocyclic Chemistry, vol. 6, 177, edited by A. Katritzky and C. W. Rees, Pergamon Press (1984).
Compounds of formula VIII (a compound of formula I where R4 is acetyl and X is sulfur) can be hydrolyzed in the presence of a base such as sodium hydroxide to provide a compound of formula IX. A compound of formula IX may then be reacted with R4xe2x80x94L, in the presence of a base such as triethylamine, where L is a leaving group such as a halogen, to give compounds of formula I where X is sulfur. In this manner, compounds of formula IX, which is a compound of formula I where R4 is hydrogen, can be treated with agents such as isothiocyanates, halides, acyl halides, chloroformates, isocyanates or sulfonyl chlorides to provide thioureas, amines, amides, carbamates, ureas or sulfonamides. The procedures in Scheme 2 specifically illustrate a methyloxazole group, but are general for all R3(CR1R2)nxe2x80x94 groups specified by formula I.
Alternatively, compounds of formula VII, where L is bromine, may be prepared by halogenation of 2-methyloxazole using N-bromosuccinimide in the presence of dibenzoylperoxide. 
Scheme 3 illustrates an alternative method of preparing compound VII, which is a compound of formula R3(CR1R2)nxe2x80x94L where L is chlorine and n is the integer 1. In this scheme, compound VII is prepared by the reaction of a compound of formula X and formula XI in the presence of a base such as triethylamine to provide compounds of formula XII. Compound XII may be oxidized by an oxidant such as oxalylchloride/DMSO in the presence of a base such as triethylamine to provide a compound of formula XIII which may be cyclized by an agent such as phosphorous oxychloride to provide compounds of formula VII, wherein L is chlorine. Alternatively, compounds of formula XIII may be prepared by reaction of the amino ketone correponding to X with an acid chloride such as XI. 
Compounds of formula VII, where L is chlorine, may also be prepared from the reaction of diazoketones as illustrated by formula XIV in Scheme 4 with chloronitriles, such as indicated by formula XV, in the presence of BF3 etherate to provide compounds of formula VII, wherein L is chlorine. 
In Scheme 5, starting compound XVI denotes a resin-bound benzyl alcohol support used for solid phase synthesis which is prepared from a Merrifield resin denoted as , and 2-methoxy-4-hydroxybenzaldehyde, followed by reduction with reducing agents such as NaBH4. In step 1, starting compound XVI is treated with triphosgene and triphenylphosphine (PPh3) in dichloromethane to give the chlorobenzyl resin of formula XVII. In step 2, a thiocyanato trifluoroacetamide (XVIII) is alkylated with the resin-bound benzyl chloride (XVII) in the presence of diisopropylethylamine (DIPEA) to form a resin-bound thiocyanate (XIX). The thiocyanato trifluoroacetamide compound of formula XVII is prepared by reacting 5-thiocyanatoaminothiazole of formula III (Scheme I) with trifluoroacetic anhydride using a base such as 2,6-lutidine.
The resin-bound thiocyanate (XIX) is then reduced to a resin-bound thiol (XX) in step 3 with reducing agent such as dithiothreitol (DTT) in tetrahydrofuran (THF) and methanol. The resulting resin-bound thiol (XX) is reacted with R3(CR1R2)nxe2x80x94L, where L is a leaving group, in the presence of a base such as 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) at 80xc2x0 C. in dimethylformamide (DMF) to form compounds of formula XXI (step 4). Deprotection of the trifluoroacetyl group of compound XXI is performed in step 5 using sodium borohydride to provide a compound of formula XII. In step 6, the deprotected compound XXII is reacted with R6X, where X is a leaving group, in the presence of a base such as diisopropylethylamine to provide compounds of formula XXIII. The product is then cleaved from the solid phase resin in step 7 with trifluoroacetic acid (TFA) to give compounds of formula I where X is sulfur. Compounds of formula I where X is S(O)m and m is 1 or 2 may be prepared from compounds of formula I where m is 0 by oxidation with an oxidant such as sodium periodate, meta-chloroperbenzoic acid, or oxone. 
Scheme 6 illustrates the preparation of compounds of formula I from a 2-bromo thiazole XXIV. A compound of formula IX is reacted with a diazotizing agent such as tBuONO in the presence of copper bromide to provide the exemplary 2-bromo thiazole of formula XXIV. Compound XXIV may then be reacted with a compound of formula R4NH2, with or without an added base, to provide compounds of formula I. 
Compounds of formula I may also be prepared starting from 2-bromothiazole XXV by reaction with a compound of formula R4NH2, with or without an added base, to provide a compound of formula XXVI. The compound of formula XXVI may be reacted with a thiocyanating agent such as sodium thiocyanate in the presence of bromine to provide a compound of formula IV, that may then be converted to a compound of formula I as described in Scheme 1. Alternatively, the compound of formula XXVI may be treated with a brominating agent such as bromine in acetic acid to generate a compound XXVII. Compounds of formula XXVII may be reacted with either XXVIII or XXIX (themselves available from a compound of formula VII) in the presence of base to provide compounds of formula I.
The starting compounds of Schemes 1-7 are commercially available or may be prepared by methods known to one of ordinary skill in the art.
All compounds of formula I may be prepared by modification of the procedures described herein.
The preferred compounds of formula I are those where:
R1 and R2 are independently hydrogen, fluorine or alkyl;
R3 is 
wherein Y is oxygen, sulfur or NR9;
R4 is alkyl, cycloalkyl, aryl, cycloalkylalkyl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, heterocycloalkylalkyl; or
CO-alkyl, CO-cycloalkyl, CO-aryl, CO-alkyl-cycloalkyl, CO-alkyl-aryl, CO-heteroaryl, CO-alkyl-heteroaryl, CO-heterocycloalkyl, CO-alkyl-heterocycloalkyl; or
CONH-alkyl, CONH-cycloalkyl, CONH-aryl, CONH-alkyl-cycloalkyl, CONH-alkyl-aryl, CONH-heteroaryl, CONH-alkyl-heteroaryl, CONH-heterocycloalkyl, CONH-alkyl-heterocycloalkyl; or
COO-alkyl, COO-cycloalkyl, COO-aryl, COO-alkyl-cycloalkyl, COO-alkyl-aryl, COO-heteroaryl, COO-alkyl-heteroaryl, COO-heterocycloalkyl, COO-alkyl-heterocycloalkyl; or
SO2-cycloalkyl, SO2-aryl, SO2-alkyl-cycloalkyl, SO2-alkyl-aryl, SO2-heteroaryl, SO2-alkyl-heteroaryl, SO2-heterocycloalkyl, SO2-alkyl-heterocycloalkyl; or
C(NCN)NH-alkyl, C(NCN)NH-cycloalkyl, C(NCN)NH-aryl, C(NCNNH)-alkyl-cycloalkyl, C(NCN)NH-alkyl-aryl, C(NCN)NH-heteroaryl, C(NCN)NH-alkyl-heteroaryl, C(NCN)NH-heterocycloalkyl, C(NCN)NH-alkyl-heterocylcoalkyl; or
C(NNO2)NH-alkyl, C(NNO2)NH-cycloalkyl, C(NNO2)NH-aryl, C(NNO2)NH-alkyl-cycloalkyl, C(NNO2)NH-alkyl-aryll C(NNO2)NH-heteroaryl, C(NNO2)NH-alkyl-heteroaryl, C(NNO2)NH-heterocyloalkyl, C(NNO2)NH-alkyl-heterocycloalkyl; or
C(NH)NH-alkyl, C(NH)NH-cycloalkyl, C(NH)NH-aryl, C(NH)NH-alkyl-cycloalkyl, C(NH)NH-alkyl-aryl, C(NH)NH-heteroaryl, C(NH)NH-alkyl-heteroaryl, C(NH)NH-heterocycloalkyl, C(NH)NH-alkyl-heterocycloalkyl; or
C(NH)NHCO-alkyl, C(NH)NHCO-cycloalkyl, C(NH)NHCO-aryl, C(NH)NHCO-alkyl-cycloalkyl, C(NH)NHCO-alkyl-aryl, C(NH)NHCO-heteroaryl, C(NH)NHCO-alkyl-heteroaryl, C(NH)NHCO-heterocylcloalkyl, C(NH)NHCO-alkyl-heterocycloalkyl; or
C(NOR6)NH-alkyl, C(NOR6)NH-cycloalkyl, C(NOR6)NH-aryl, C(NOR6)NH-alkyl-cycloalkyl, C(NOR6)NH-alkyl-aryl, C(NOR6)NH-heteroaryl, C(NOR6)NH-alkyl-heteroaryl, C(NOR6)NH-heterocylcoalkyl, C(NOR6)NH-alkyl-heterocycloalkyl;
R5 is hydrogen; and
R6 is hydrogen, alkyl, cycloalkyl, aryl, cycloalkylakyl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl or heterocycloalkylalkyl;
R7 and R8 are independently hydrogen, alkyl, cycloalkyl, aryl, alkylcycloalkyl, alkylaryl, heteroaryl, alkylheteroaryl, heterocycloalkyl, alkylheterocycloalkyl or halogen;
R9 is H or alkyl;
m is the integer 0; and
n is the integer 1.
The most preferred compounds of formula I are those where:
R1 is hydrogen;
R2 is hydrogen, fluorine or alkyl;
R3 is a substituted oxazole having the configuration: 
R4 is CO-alkyl, CO-alkyl-aryl, CO-cycloalkyl, CO-alkyl-heteroaryl, CO-alkyl-heteroalkyl, CO-alkyl-heterocycloalkyl, aryl, arylalkyl, heteroaryl,
heteroarylalkyl;
R5 is hydrogen;
R7 is hydrogen;
R8 is an alkyl group, such as tert-butyl;
m is the integer 0; and
n is the integer 1.
The compounds according to the invention have pharmacological properties; in particular, the compounds of formula I are inhibitors of protein kinases such as the cyclin dependent kinases (cdks), for example, cdc2 (cdk1), cdk2, and cdk4. The novel compounds of formula I are expected to be useful in the therapy of proliferative diseases such as cancer, autoimmune diseases, viral diseases, fungal diseases, neurodegenerative disorders and cardiovascular disease.
More specifically, the compounds of formula I are useful in the treatment of a variety of cancers, including (but not limited to) the following:
carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, esophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma;
hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burkett""s lymphoma;
hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia;
tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma;
tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; and
other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi""s sarcoma.
Due to the key role of cdks in the regulation of cellular proliferation in general, inhibitors could act as reversible cytostatic agents which may be useful in the treatment of any disease process which features abnormal cellular proliferation, e.g., benign prostate hyperplasia, familial adenomatosis polyposis, neuro-fibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, hypertrophic scar formation, inflammatory bowel disease, transplantation rejection, endotoxic shock, and fungal infections.
Compounds of formula I may also be useful in the treatment of Alzheimer""s disease, as suggested by the recent finding that cdk5 is involved in the phosphorylation of tau protein (J. Biochem, 117, 741-749 (1995)).
Compounds of formula I may induce or inhibit apoptosis. The apoptotic response is aberrant in a variety of human diseases. Compounds of formula I, as modulators of apoptosis, will be useful in the treatment of cancer (including but not limited to those types mentioned hereinabove), viral infections (including but not limited to herpevirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus), prevention of AIDS development in HIV-infected individuals, autoimmune diseases (including but not limited to systemic lupus, erythematosus, autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes mellitus), neurodegenerative disorders (including but not limited to Alzheimer""s disease, AIDS-related dementia, Parkinson""s disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration), myelodysplastic syndromes, aplastic anemia, ischemic injury associated with myocardial infarctions, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol related liver diseases, hematological diseases (including but not limited to chronic anemia and aplastic anemia), degenerative diseases of the musculoskeletal system (including but not limited to osteoporosis and arthritis) aspirin-sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis, kidney diseases and cancer pain.
Compounds of formula I, as inhibitors of the cdks, can modulate the level of cellular RNA and DNA synthesis. These agents would therefore be useful in the treatment of viral infections (including but not limited to HIV, human papilloma virus, herpesvirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus).
Compounds of formula I may also be useful in the chemoprevention of cancer. Chemoprevention is defined as inhibiting the development of invasive cancer by either blocking the initiating mutagenic event or by blocking the progression of pre-malignant cells that have already suffered an insult or inhibiting tumor relapse.
Compounds of formula I may also be useful in inhibiting tumor angiogenesis and metastasis.
Compounds of formula I may also act as inhibitors of other protein kinases, e.g., protein kinase C, her2, raf1, MEK1, MAP kinase, EGF receptor, PDGF receptor, IGF receptor, PI3 kinase, weel kinase, Src, Abl and thus be effective in the treatment of diseases associated with other protein kinases.
The compounds of this invention may also be useful in combination (administered together or sequentially) with known anti-cancer treatments such as radiation therapy or with cytostatic or cytotoxic agents, such as for example, but not limited to, DNA interactive agents, such as cisplatin or doxorubicin; topoisomerase II inhibitors, such as etoposide; topoisomerase I inhibitors such as CPT-11 or topotecan; tubulin interacting agents, such as paclitaxel, docetaxel or the epothilones; hormonal agents, such as tamoxifen; thymidilate synthase inhibitors, such as 5-fluorouracil; and anti-metabolites, such as methoxtrexate. Compounds of formula I may also be useful in combination with modulators of p53 transactivation.
If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described below and the other pharmaceutically active agent or treatment within its approved dosage range. For example, the cdc2 inhibitor olomucine has been found to act synergistically with known cytotoxic agents in inducing apoptosis (J. Cell Sci., 108, 2897 (1995)). Compounds of formula I may also be administered sequentially with known anticancer or cytotoxic agents when a combination formulation is inappropriate. The invention is not limited in the sequence of administration; compounds of formula I may be administered either prior to or after administration of the known anticancer or cytotoxic agent. For example, the cytotoxic activity of the cyclin-dependent kinase inhibitor flavopiridol is affected by the sequence of administration with anticancer agents. Cancer Research, 57, 3375 (1997).
The pharmacological properties of the compounds of this invention may be confirmed by a number of pharmacological assays. The exemplified pharmacological assays which follow have been carried out with the compounds according to the invention and their salts. The compounds of examples 1 to 14 exhibited cdc2/cyclin B1 kinase activity with IC50 values less than 50 xcexcM. The compounds of examples 1 to 14 exhibited cdk2/cyclin E kinase activity with IC50 values less than 50 xcexcM. The compounds of examples 1 to 14 exhibited cdk4/cyclin D1 kinase activity with IC50 values less than 50 xcexcM.
cdc2/cyclin B1 kinase activity was determined by monitoring the incorporation of 32P into histone H1. The reaction consisted of 50 ng baculovirus expressed GST-cdc2, 75 ng baculovirus expressed GST-cyclin B1, 1 xcexcg histone HI (Boehringer Mannheim), 0.2 mCi of 32P g-ATP and 25 mM ATP in kinase buffer (50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 0.5 mM DTT). The reaction was incubated at 30xc2x0 C. for 30 minutes and then stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration of 15% and incubated on ice for 20 minutes. The reaction was harvested onto GF/C unifilter plates (Packard) using a Packard Filtermate Universal harvester, and the filters were counted on a Packard TopCount 96-well liquid scintillation counter (Marshak, D. R., Vanderberg, M. T., Bae, Y. S., Yu, I. J., J. of Cellular Biochemistry, 45, 391-400 (1991), incorporated by reference herein).
cdk2/cyclin E kinase activity was determined by monitoring the incorporation of 32P into the retinoblastoma protein. The reaction consisted of 2.5 ng baculovirus expressed GST-cdk2/cyclin E, 500 ng bacterially produced GST-retinoblastoma protein (aa 776-928), 0.2 mCi 32P g-ATP and 25 mM ATP in kinase buffer (50 mM Hepes, pH 8.0, 10 mM MgCl2, 5 mM EGTA, 2 mM DTT). The reaction was incubated at 30xc2x0 C. for 30 minutes and then stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration of 15% and incubated on ice for 20 minutes. The reaction was harvested onto GF/C unifilter plates (Packard) using a Packard Filtermate Universal harvester, and thefilters were counted on a Packard TopCount 96-well liquid scintillation counter.
cdk4/cyclin D1 kinase activity was determined by monitoring the incorporation of 32P in to the retinoblastoma protein. The reaction consisted of 165 ng baculovirus expressed as GST-cdk4, 282 ng bacterially expressed as S-tag cyclin D1, 500 ng bacterially produced GST-retinoblastoma protein (aa 776-928), 0.2 xcexcCi 32P xcex3-ATP and 25 xcexcm ATP in kinase buffer (50 mM Hepes, pH 8.0, 10 mM MgCl2, 5 mM EGTA, 2 mM DTT). The reaction was incubated at 30xc2x0 C. for 1 hour and then stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration of 15% and incubated on ice for 20 minutes. The reaction was harvested onto GF/C unifilter plates (Packard) using a Packard Filtermate Universal harvester, and the filters were counted on a Packard TopCount 96-well liquid scintillation counter (Coleman, K. G., Wautlet, B. S., Morissey, D, Mulheron, J. G., Sedman, S., Brinkley, P., Price, S., Wedster, K. R. (1997). Identification of CDK4 Sequences involved in cyclin D, and p16 binding. J. Biol. Chem. 272,30:18869-18874, incorporated by reference herein).
Further subject matter of the invention also includes pharmaceuticals for use as described above including controlling cancer, inflammation and arthritis, which contain at least one compound of the formula I as defined above or at least one of its pharmacologically acceptable acid addition salts, and the use of a compound of the formula I as defined above for the preparation of a pharmaceutical having activity against proliferative diseases as described previously including against cancer, inflammation and/or arthritis.
The following examples and preparations describe the manner and process of making and using the invention and are illustrative rather than limiting. It should be understood that there may be other embodiments which fall within the spirit and scope of the invention as defined by the claims appended hereto.